PROJECT SUMMARY Asthma is an episodic inflammatory disease that affects over 25 million Americans and manifests as airway hyperresponsiveness to specific environmental stimuli. Despite effective medications that control asthma in most individuals, 15% respond inadequately and suffer life-threatening exacerbations. A feature shared by most patients with severe disease is glucocorticoid insensitivity, a poorly understood physiological process. The airway smooth muscle (ASM), which plays an important role in asthma, is a target of glucocorticoids that act in part via modulation of gene transcription, alteration of histone post-translational modifications, and inhibition of transcription factors. CCAAT/Enhancer Binding Protein D (CEBPD) is a pleiotropic glucocorticoid- responsive transcription factor that regulates inflammatory responses, cell differentiation and tissue remodeling. Based on our data showing that CEBPD expression is (1) lower in fatal asthma vs. non-asthma ASM, (2) induced with glucocorticoid treatment in non-asthma ASM but unchanged in fatal asthma ASM, and that (3) decreasing CEBPD via knockdown resulted in increased IL1?-induced NFkB-luciferase expression with glucocorticoid stimulation, our central hypothesis states that low CEBPD levels in ASM elicits transcriptomic and epigenetic modifications that decrease glucocorticoid sensitivity. We will test this hypothesis by using the following unbiased and complementary `omic approaches to study the effect of CEBPD on glucocorticoid response in ASM from fatal asthma and non-asthma donors: (1) RNA-Seq to identify transcriptomic effects, (2) proteomics to determine global histone post-translational modification effects, and (3) ChIP-Seq to measure transcription factor binding of NFkB and the glucocorticoid receptor. An integrated analysis of RNA-Seq, proteomics and ChIP-Seq results will identify major targets of CEBPD that influence glucocorticoid response, whose role will be confirmed via NFkB-luciferase assays. Our project will offer insights into transcriptional and epigenetic signatures that are characteristic of severe asthma, enable glucocorticoid sensitivity biomarker development, and offer therapeutic insights that benefit the most vulnerable individuals with asthma.